Vaccine against rsv

ABSTRACT

Provided is a vaccine against respiratory syncytial virus (RSV), comprising a recombinant human adenovirus of serotype  26  that comprises nucleic acid molecule encoding a RSV F protein or immunologically active part thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. patent applicationSer. No. 13/849,380, filed Mar. 22, 2013, now U.S. Pat. No. ______,which application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/614,429, filed Mar. 22, 2012, the disclosure ofeach of which is hereby incorporated herein in its entirety by thisreference.

TECHNICAL FIELD

The application relates to the fields of biotechnology and medicine,particularly it relates to vaccines against respiratory syncytial virus(RSV).

BACKGROUND

After discovery of RSV in the 1950s, the virus soon became a recognizedpathogen associated with lower and upper respiratory tract infections inhumans. Worldwide, it is estimated that 64 million RSV infections occureach year resulting in 160,000 deaths (WHO Acute Respiratory InfectionsUpdate September 2009). The most severe disease occurs particularly inpremature infants, the elderly and immunocompromised individuals. Inchildren younger than 2 years, RSV is the most common respiratory tractpathogen, accounting for approximately 50% of the hospitalizations dueto respiratory infections, and the peak of hospitalization occurs at 2-4months of age. It has been reported that almost all children have beeninfected by RSV by the age of two. Repeated infection during lifetime isattributed to ineffective natural immunity. The level of RSV diseaseburden, mortality and morbidity in the elderly are second to thosecaused by nonpandemic influenza A infections.

RSV is a paramyxovirus, belonging to the subfamily of pneumovirinae. Itsgenome encodes for various proteins, including the membrane proteinsknown as RSV Glycoprotein (G) and RSV fusion (F) protein which are themajor antigenic targets for neutralizing antibodies. Proteolyticcleavage of the fusion protein precursor (FO) yields two polypeptides Fland F2 linked via disulfide bridge. Antibodies against thefusion-mediating part of the F1 protein can prevent virus uptake in thecell and thus have a neutralizing effect. Besides being a target forneutralizing antibodies, RSV F contains cytotoxic T cell epitopes(Pemberton et al., 1987, J. Gen. Virol. 68: 2177-2182).

Treatment options for RSV infection include a monoclonal antibodyagainst the F protein of RSV. The high costs associated with suchmonoclonal antibodies and the requirement for administration in ahospital setting; preclude their use for prophylaxis in the at-riskpopulation at large scale. Thus, there is a need for an RSV vaccine,which preferably can be used for the pediatric population as well as forthe elderly.

Despite 50 years of research, there is still no licensed vaccine againstRSV. One major obstacle to the vaccine development is the legacy ofvaccine-enhanced disease in a clinical trial in the 1960s with aformalin-inactivated (FI) RSV vaccine. FI-RSV vaccinated children werenot protected against natural infection and infected childrenexperienced more severe illness than non-vaccinated children, includingtwo deaths. This phenomenon is referred to as “enhanced disease.”

Since the trial with the FI-RSV vaccine, various approaches to generatean RSV vaccine have been pursued. Attempts include classical liveattenuated cold passaged or temperature-sensitive mutant strains of RSV,(chimeric) protein subunit vaccines, peptide vaccines and RSV proteinsexpressed from recombinant viral vectors. Although some of thesevaccines showed promising pre-clinical data, no vaccine has beenlicensed for human use due to safety concerns or lack of efficacy.

Adenovirus vectors are used for the preparation of vaccines for avariety of diseases, including disease associated with RSV infections.The following paragraphs provide examples of adenovirus-based RSVcandidate vaccines that have been described.

In one approach, RSV.F has been inserted into the non-essential E3region of replication competent adenovirus types 4, 5, and 7.Immunization in cotton rats, intranasal (i.n.) application of 10⁷ pfu,was moderately immunogenic, and protective against lower respiratorytracts against RSV challenge, but not protective against upperrespiratory tract RSV challenge (Connors et al., 1992, Vaccine10:475-484; P. L. Collins, G. A. Prince, E. Camargo, R. H. Purcell, R.M. Chanock, and B. R. Murphy, evaluation of the protective efficacy ofrecombinant vaccinia viruses and adenoviruses that express respiratorysyncytial virus glycoproteins, in Vaccines 90: Modern Approaches to NewVaccines including prevention of AIDS (Eds. F. Brown, R. M. Chanock, H.Ginsberg, and R. A. Lerner) Cold Spring Harbor Laboratory, New York,1990, pp 79-84). Subsequent oral immunization of a chimpanzee was poorlyimmunogenic (Hsu et al., 1992, J. Infect. Dis. 66:769-775).

In other studies (Shao et al., 2009, Vaccine 27:5460-71; U.S.2011/0014220), two recombinant replication incompetent adenovirus 5vectors carrying nucleic acid molecule encoding the transmembranetruncated (rAd-F0ΔTM) or full-length (rAd-F0) version of the F proteinof the RSV-B1 strain were engineered and given via the intranasal routeto BALB/c mice. Animals were primed i.n. with 10⁷ pfu and boosted 28days later with the same dose i.n. Although the anti-RSV-B1 antibodieswere neutralizing and cross-reacting with RSV-Long and RSV-A2 strain,immunization with these vectors protected only partially against RSV B1challenge replication. The (partial) protection with rAd-F0ΔTM wasslightly higher than with rAd-F0.

In another study, it was observed that BALB/c mice i.n. immunizationwith 10¹¹ virus particles with the replication deficient (Ad5-based)FG-Ad adenovirus expressing wild-type RSV F (FG-Ad-F) reduced lung viraltiters only a 1.5 log 10 compared with the control group (Fu et al.,2009, Biochem. Biophys. Res. Commun. 381:528-532.

In yet other studies, it was observed that intranasally appliedrecombinant Ad5-based replication-deficient adenovector expressing codonoptimized soluble F1 fragment of F protein of RSV A2 (amino acid155-524) (10⁸ PFU) could reduce RSV challenge replication in the lungsof BALB/c mice compared to control mice, but mice immunized by theintramuscular (i.m.) route did not exhibit any protection from thechallenge (Kim et al., 2010, Vaccine 28:3801-3808).

In other studies, adenovectors Ad5-based carrying the codon optimizedfull-length RSV F (AdV-F) or the soluble form of the RSF F gene(AdV-Fsol) were used to immunize BALB/c mice twice with a dose of 1×10¹⁰OPU (optical particle units: a dose of 1×10¹⁰ OPU corresponds with 2×10⁸GTU (gene transduction unit)). These vectors strongly reduced viralloads in the lungs after i.n. immunization, but only partially aftersubcutaneous (s.c.) or i.m. application (Kohlmann et al., 2009, J.Virol. 83:12601-12610; U.S. 2010/0111989).

In yet other studies, it was observed that intramuscular appliedrecombinant Ad5-based replication-deficient adenovector expressing thesequenced F protein cDNA of RSV A2 strain (10¹⁰ particle units) couldreduce RSV challenge replication only partially in the lungs of BALB/cmice compared to control mice (Krause et al., 2011, Virology Journal8:375-386).

Apart from not being fully effective in many cases, the RSV vaccinesunder clinical evaluation for pediatric use and most of the vaccinesunder pre-clinical evaluation are intranasal vaccines. The mostimportant advantages of the intranasal strategy are the directstimulation of local respiratory tract immunity and the lack ofassociated disease enhancement. Indeed, generally the efficacy of, forinstance, the adenovirus-based RSV candidate vaccines appears better forintranasal administration as compared to intramuscular administration.However, intranasal vaccination also gives rise to safety concerns ininfants younger than 6 months. Most common adverse reactions ofintranasal vaccines are runny nose or nasal congestion in all ages.Newborn infants are obligate nasal breathers and thus must breathethrough the nose. Therefore, nasal congestion in an infant's first fewmonths of life can interfere with nursing, and in rare cases can causeserious breathing problems.

More than 50 different human adenovirus serotypes have been identified.Of these, adenovirus serotype 5 (Ad5) has historically been studied mostextensively for use as gene carrier. Recombinant adenoviral vectors ofdifferent serotypes may however give rise to different results withrespect to induction of immune responses and protection. For instance,WO 2012/021730 describes that simian adenoviral vector serotype 7 andhuman adenoviral vector serotype 5 encoding F protein provide betterprotection against RSV than a human adenoviral vector of serotype 28. Inaddition, differential immunogenicity was observed for vectors based onhuman or non-human adenovirus serotypes (Abbink et al., 2007, J. Virol.81:4654-4663; Colloca et al., 2012, Sci. Transl. Med. 4, 115ra2). Abbinket al., conclude that all rare serotype human rAd vectors studied wereless potent than rAd5 vectors in the absence of anti-Ad5 immunity.Further it has been recently described that, while rAd5 with anEbolavirus (EBOV) glycoprotein (gp) transgene protected 100% ofnon-human primates, rAd35 and rAd26 with EBOV gp transgene provided onlypartial protection and a heterologous prime-boost strategy was requiredwith these vectors to obtain full protection against ebola viruschallenge (Geisbert et al., 2011, J. Virol. 85:4222-4233). Thus, it is apriori not possible to predict the efficacy of a recombinant adenoviralvaccine, based solely on data from another adenovirus serotype.

Moreover, for RSV vaccines, experiments in appropriate disease modelssuch as cotton rat are required to determine if a vaccine candidate isefficacious enough to prevent replication of RSV in the nasal tract andlungs and at the same time is safe, i.e., does not lead to enhanceddisease. Preferably such candidate vaccines should be highly efficaciousin such models, even upon intramuscular administration.

SUMMARY OF THE DISCLOSURE

It was surprisingly found by the present inventors that recombinantadenoviruses of serotype 26 (Ad26) that comprise a nucleotide sequenceencoding RSV F protein are very effective vaccines against RSV in a wellestablished cotton rat model, and have improved efficacy as compared todata described earlier for Ad5 encoding RSV F. It is demonstrated thateven a single administration, even intramuscularly, of Ad26 encoding RSVF is sufficient to provide complete protection against challenge RSVreplication.

The vaccines hereof based on Ad26 surprisingly appear more potent thanthe ones described in the prior art that were based upon Ad5, since theAd5-based vaccines failed to provide complete protection against RSVchallenge replication after a single intramuscular administration.

Provided is a vaccine against respiratory syncytial virus (RSV),comprising a recombinant human adenovirus of serotype 26 or 35 thatcomprises nucleic acid molecule encoding a RSV F protein or fragmentthereof.

In certain embodiments, the recombinant adenovirus comprises a nucleicacid molecule encoding RSV F protein comprising SEQ ID NO:1 of theincorporated herein Sequence Listing.

In certain embodiments, the nucleic acid molecule encoding RSV F proteinis codon optimized for expression in human cells. In such embodiments,the nucleic acid molecule encoding RSV F protein may comprise SEQ IDNO:2.

In certain embodiments, the recombinant human adenovirus has a deletionin the El region, a deletion in the E3 region, or a deletion in both theE1 and the E3 regions of the adenoviral genome.

In certain embodiments, the recombinant adenovirus has a genomecomprising, at its 5′ terminal ends, the polynucleotide CTATCTAT.

Further provided is a method for vaccinating a subject against RSV, themethod comprising administering to the subject a vaccine hereof.

In certain embodiments, the vaccine is administered intramuscularly.

In certain embodiments, a vaccine hereof is administered to the subjectmore than once.

In certain embodiments, the method for vaccinating a subject against RSVfurther comprises administering to the subject a vaccine comprising arecombinant human adenovirus of serotype 35 that comprises nucleic acidmolecule encoding a RSV F protein or fragment thereof.

In certain embodiments, the method of vaccinating a subject against RSVfurther comprises administering RSV F protein (preferably formulated asa pharmaceutical composition, thus, a protein vaccine) to the subject.

In certain embodiments, the method for vaccination consists of a singleadministration of the vaccine to the subject.

Also provided is a method for reducing infection and/or replication ofRSV in, e.g., the nasal tract and lungs of, a subject, the methodcomprising administering to the subject by intramuscular injection of acomposition comprising a recombinant human adenovirus of serotype 26comprising nucleic acid molecule encoding a RSV F protein or fragmentthereof. This administration will reduce adverse effects resulting fromRSV infection in a subject, and thus contribute to protection of thesubject against such adverse effects upon administration of the vaccine.In certain embodiments, adverse effects of RSV infection may beessentially prevented, i.e., reduced to such low levels that they arenot clinically relevant. The recombinant adenovirus may be in the formof a vaccine hereof, including the embodiments described above.

Also provided is an isolated host cell comprising a recombinant humanadenovirus of serotype 26 comprising nucleic acid molecule encoding aRSV F protein or fragment thereof

Further provided is a method for making a vaccine against respiratorysyncytial virus (RSV), comprising providing a recombinant humanadenovirus of serotype 26 that comprises nucleic acid molecule encodinga RSV F protein or fragment thereof, propagating said recombinantadenovirus in a culture of host cells, isolating and purifying therecombinant adenovirus, and formulating the recombinant adenovirus in apharmaceutically acceptable composition. The recombinant humanadenovirus of this aspect may also be any of the adenoviruses describedin the embodiments above.

Also provided is an isolated recombinant nucleic acid molecule thatforms the genome of a recombinant human adenovirus of serotype 26 thatcomprises nucleic acid molecule encoding a RSV F protein or fragmentthereof The adenovirus may also be any of the adenoviruses as describedin the embodiments above.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the cellular immune response against F peptidesoverlapping the aa 1-252 of F and F peptides overlapping the aa 241-574of F of mice upon immunization with different doses of rAd26-(FIG. 1A)and rAd35-(FIG. 1B) based vectors harboring the RSV F gene at 2 and 8weeks after immunization.

FIG. 2 shows the antibody response against RSV in mice upon immunizationwith different doses of rAd26- and rAd35-based vectors harboring the RSVF gene at 2 and 8 weeks after immunization.

FIG. 3 shows the results of ratio of IgG2a vs. IgG 1 antibody responseagainst RSV in mice upon immunization with 10¹⁰ vp of rAd26- andrAd35-based vectors harboring the RSV F gene at 8 weeks afterimmunization.

FIG. 4 shows the virus neutralization capacity against RSV Long in miceupon immunization with different doses of rAd26-(A) and rAd35-(B) basedvectors harboring the RSV F gene at 2 and 8 weeks after immunization.

FIGS. 5A and 5B show the cellular immune response against (FIG. 5A) Fpeptides overlapping the aa 1-252 of F and (FIG. 5B) F peptidesoverlapping the aa 241-574 of F of mice upon prime boost immunizationwith rAd26- and rAd35-based vectors harboring the RSV F gene at 6 and 12weeks after primary immunization.

FIG. 6 shows the antibody response against RSV in mice upon prime boostimmunization with rAd26- and rAd35-based vectors harboring the RSV Fgene at different time points after the first immunization.

FIG. 7 shows the virus neutralization capacity against RSV Long in miceserum upon prime boost immunization with different doses of rAd26- andrAd35-based vectors harboring the RSV F gene at different time pointsafter the first immunization.

FIG. 8 shows the virus neutralization capacity against RSV B1 in miceupon prime boost immunization with different doses of rAd26- andrAd35-based vectors harboring the RSV F gene at different time pointsafter the first immunization.

FIG. 9 shows the A) RSV lung titers and B) RSV nose titers in the cottonrats following prime boost immunization with different doses of rAd26-and rAd35-based vectors harboring the RSV F gene at 5 days postchallenge.

FIG. 10 shows the induction of virus neutralizing titers following primeboost immunization with different doses of rAd26- and rAd35-basedvectors harboring the RSV F gene at A) 28 days, and B) 49 days after thefirst immunization.

FIG. 11 shows the histopathological examination of the cotton rat lungsat day of sacrifice following prime boost immunization with differentdoses of rAd26- and rAd35-based vectors harboring the RSV F gene.

FIG. 12 shows A) the RSV lung titers and B) the RSV nose titers in thecotton rats following single dose immunization with different doses ofrAd26- and rAd35-based vectors harboring the RSV F gene at 5 days postchallenge, administered via different routes.

FIG. 13 shows the induced virus neutralizing titers following singledose immunization with different doses of rAd26- and rAd35-based vectorsharboring the RSV F gene at 28 and 49 days after the first immunization,administered via different routes.

FIG. 14 shows the histopathological examination of the cotton rat lungsat day of sacrifice following single dose immunization (i.m.) withdifferent doses of rAd26- and rAd35-based vectors harboring the RSV Fgene at day of sacrifice.

FIG. 15 shows maps of plasmids comprising the left end of the genome ofAd35 and Ad26 with the sequence encoding RSV F: A.pAdApt35BSU.RSV.F(A2)nat, and B. pAdApt26.RSV.F(A2)nat.

FIG. 16 shows A) the RSV lung titers and B) the RSV nose titers in thecotton rats following single dose immunization at day 0 or day 28 withdifferent doses of rAd26-based vectors harboring the RSV F gene at 5days post challenge. Challenge was at day 49.

FIG. 17 shows the induction of virus neutralizing titers followingsingle dose immunization with different doses of rAd26 harboring the RSVF gene at 49 days after immunization as described for FIG. 16.

FIG. 18 shows the induction of virus neutralizing titers followingsingle dose immunization with different doses of rAd26 harboring the RSVF gene during time after immunization.

FIG. 19 shows the VNA titers 49 days after against RSV Long and RSVBwash with serum derived from cotton rats immunized with 10¹⁰ of Ad-RSVF or no transgene (Ad-e). PB: prime boost.

FIG. 20 shows the RSV lung titers in the cotton rats following singledose immunization at day 0 with different doses of rAd26-based vectorsharboring the RSV F gene at 5 days post challenge with RSV A2 or RSVB15/97.

FIG. 21 shows the RSV nose titers in the cotton rats following singledose immunization at day 0 with different doses of rAd26-based vectorsharboring the RSV F gene at 5 days post challenge with RSV A2 or RSVB15/97.

FIG. 22 shows the VNA titers in cotton rat serums following single doseimmunization at day 0 with different doses of rAd26-based vectorsharboring the RSV F gene at different time points post prime.

FIG. 23 shows the RSV lung titers in the cotton rats following singledose immunization at day 0 with different doses of rAd26-based vectorsharboring the RSV F gene at 5 days post challenge with a standard dose(10⁵) or a high dose (5×10⁵) of RSV A2.

FIG. 24 shows the RSV nose titers in the cotton rats following singledose immunization at day 0 with different doses of rAd26-based vectorsharboring the RSV F gene at 5 days post challenge with challenge with astandard dose (10⁵) or a high dose (5×10⁵) of RSV A2.

FIG. 25 shows the RSV lung titers in the cotton rats followingimmunization at day 0 and 28 with different doses of single immunizationor prime boost immunization with rAd26-based vectors harboring the RSV Fgene at 5 days post challenge, with the challenge performed 210 dayspost immunization.

FIG. 26 shows the VNA titers of the cotton rat serum following singledose and prime boost immunization at 140 days post immunization.

FIG. 27 shows the histopathological examination of the cotton rat lungsof sacrifice following single immunization or prime boost immunizationwith different doses of rAd26-based vectors harboring the RSV F gene at2 days post challenge. Dots represent the median and whiskers the25^(th) and 75^(th) percentile.

FIG. 28 shows the histopathological examination of the cotton rat lungsof sacrifice following single immunization or prime boost immunizationwith different doses of rAd26-based vectors harboring the RSV F gene at6 days post challenge. Dots represent the median and whiskers the25^(th) and 75^(th) percentile.

FIG. 29 shows the induction of virus neutralizing titers followingimmunization with rAd26 harboring the RSV F gene (Ad26.RSV.F) followedby boosting with Ad26.RSV.F or with adjuvanted RSV F protein (post-F).

FIG. 30 shows the induction of IgG2a and IgG1 antibodies, and the ratiohereof, following immunization with Ad26.RSV.F followed by boosting withAd26.RSV.F or by boosting with adjuvanted RSV F protein (post-F).

FIG. 31 shows the production of IFN-g by splenocytes followingimmunization with Ad26.RSV.F followed by boosting with Ad26.RSV.F orwith adjuvanted RSV F protein (post-F).

DETAILED DESCRIPTION

The term “recombinant” for an adenovirus, as used herein implicates thatit has been modified by the hand of man, e.g., it has altered terminalends actively cloned therein and/or it comprises a heterologous gene,i.e., it is not a naturally occurring wild-type adenovirus.

Sequences herein are provided from 5′ to 3′ direction, as custom in theart.

An “adenovirus capsid protein” refers to a protein on the capsid of anadenovirus that is involved in determining the serotype and/or tropismof a particular adenovirus. Adenoviral capsid proteins typically includethe fiber, penton and/or hexon proteins. An adenovirus of (or “basedupon”) a certain serotype hereof typically comprises fiber, pentonand/or hexon proteins of that certain serotype, and preferably comprisesfiber, penton and hexon protein of that certain serotype. These proteinsare typically encoded by the genome of the recombinant adenovirus. Arecombinant adenovirus of a certain serotype may optionally compriseand/or encode other proteins from other adenovirus serotypes. Thus, asnon-limiting example, a recombinant adenovirus that comprises hexon,penton and fiber of Ad26 is considered a recombinant adenovirus basedupon Ad26.

A recombinant adenovirus is “based upon” an adenovirus as used herein,by derivation from the wild-type, at least in sequence. This can beaccomplished by molecular cloning, using the wild-type genome or partsthereof as starting material. It is also possible to use the knownsequence of a wild-type adenovirus genome to generate (parts of) thegenome de novo by DNA synthesis, which can be performed using routineprocedures by service companies having business in the field of DNAsynthesis and/or molecular cloning (e.g., GeneArt, Invitrogen,GenScripts, Eurofins).

It is understood by a skilled person that numerous differentpolynucleotides and nucleic acid molecules can encode the samepolypeptide as a result of the degeneracy of the genetic code. It isalso understood that skilled persons may, using routine techniques, makenucleotide substitutions that do not affect the polypeptide sequenceencoded by the polynucleotides described there to reflect the codonusage of any particular host organism in which the polypeptides are tobe expressed. Therefore, unless otherwise specified, a “nucleotidesequence encoding an amino acid sequence” includes all nucleotidesequences that are degenerate versions of each other and that encode thesame amino acid sequence. Polynucleotides that encode proteins and RNAmay include introns.

In certain embodiments, the nucleic acid molecule encoding the RSV Fprotein or fragment thereof is codon optimized for expression inmammalian cells, such as human cells. Methods of codon-optimization areknown and have been described previously (e.g., WO 96/09378). An exampleof a specific codon-optimized sequence of RSV F protein is described inSEQ ID NO:2 of EP 2102345 B1.

In one embodiment, the RSV F protein is from an RSV A2 strain, and hasthe amino acid sequence of SEQ ID NO:1. In a particularly preferredembodiment, the nucleic acid molecule encoding the RSV F proteincomprises the nucleic acid molecule sequence of SEQ ID NO:2. It wasfound by the inventors that this embodiment results in stable expressionand that a vaccine according to this embodiment provides protection toRSV replication in the nasal tract and lungs even after a single dosethat was administered intramuscularly.

The term “fragment” as used herein refers to a peptide that has anamino-terminal and/or carboxy-terminal and/or internal deletion, butwhere the remaining amino acid sequence is identical to thecorresponding positions in the sequence of a RSV F protein, for example,the full-length sequence of a RSV F protein. It will be appreciated thatfor inducing an immune response and in general for vaccination purposes,a protein needs not to be full length nor have all its wild-typefunctions, and fragments of the protein are equally useful. Indeed,fragments of RSV F protein like F1 or F soluble have been shown to beefficacious in inducing immune responses like full-length F (Shao etal., 2009, Vaccine 27:5460-71, Kohlmann et al., 2009, J. Virol.83:12601-12610). Incorporation of F-protein fragments corresponding tothe amino acids 255-278 or 412-524 into active immunization induceneutralizing antibodies and some protection against RSV challenge (Singet al., 2007, Virol. Immunol. 20, 261-275; Sing et al., 2007, Vaccine25, 6211-6223).

A fragment hereof is an immunologically active fragment, and typicallycomprises at least 15 amino acids, or at least 30 amino acids, of theRSV F protein. In certain embodiments, it comprises at least 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of theRSV F protein.

The person skilled in the art will also appreciate that changes can bemade to a protein, e.g., by amino acid substitutions, deletions,additions, etc., e.g., using routine molecular biology procedures.Generally, conservative amino acid substitutions may be applied withoutloss of function or immunogenicity of a polypeptide. This can easily bechecked according to routine procedures well known to the skilledperson.

The term “vaccine” refers to an agent or composition containing anactive component effective to induce a therapeutic degree of immunity ina subject against a certain pathogen or disease. The vaccine comprisesan effective amount of a recombinant adenovirus that encodes an RSV Fprotein, or an antigenic fragment thereof, which results in an immuneresponse against the F protein of RSV. This provides a method ofpreventing serious lower respiratory tract disease leading tohospitalization and the decrease the frequency of complications such aspneumonia and bronchiolitis due to RSV infection and replication in asubject. Thus, also provided is a method for preventing or reducingserious lower respiratory tract disease, preventing or reducing (e.g.,shortening) hospitalization, and/or reducing the frequency and/orseverity of pneumonia or bronchiolitis caused by RSV in a subject,comprising administering to the subject by intramuscular injection of acomposition comprising a recombinant human adenovirus of serotype 26comprising nucleic acid molecule encoding a RSV F protein or fragmentthereof. The term “vaccine” hereof implies that it is a pharmaceuticalcomposition, and thus typically includes a pharmaceutically acceptablediluent, carrier or excipient. It may or may not comprise further activeingredients. In certain embodiments it may be a combination vaccine thatfurther comprises other components that induce an immune response, e.g.,against other proteins of RSV and/or against other infectious agents.

The vectors hereof are recombinant adenoviruses, also referred to asrecombinant adenoviral vectors. The preparation of recombinantadenoviral vectors is well known in the art.

In certain embodiments, an adenoviral vector hereof is deficient in atleast one essential gene function of the El region, e.g., the E1a regionand/or the E1b region, of the adenoviral genome that is required forviral replication. In certain embodiments, an adenoviral vector hereofis deficient in at least part of the non-essential E3 region. In certainembodiments, the vector is deficient in at least one essential genefunction of the E1 region and at least part of the non-essential E3region. The adenoviral vector can be “multiply deficient,” meaning thatthe adenoviral vector is deficient in one or more essential genefunctions in each of two or more regions of the adenoviral genome. Forexample, the aforementioned E1-deficient or E1-, E3-deficient adenoviralvectors can be further deficient in at least one essential gene of theE4 region and/or at least one essential gene of the E2 region (e.g., theE2A region and/or E2B region).

Adenoviral vectors, methods for construction thereof and methods forpropagating thereof, are well known in the art and are described in, forexample, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806,5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and6,113,913, and Thomas Shenk, “Adenoviridae and their Replication,” M. S.Horwitz, “Adenoviruses,” Chapters 67 and 68, respectively, in Virology,B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996),and other references mentioned herein. Typically, construction ofadenoviral vectors involves the use of standard molecular biologicaltechniques, such as those described in, for example, Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989); Watson et al., Recombinant DNA,2d ed., Scientific American Books (1992); and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers, N.Y.(1995), and other references mentioned herein.

An adenovirus may be a human adenovirus of the serotype 26. The vaccineshereof based on this serotype as well as those based on Ad35surprisingly appear more potent than the ones described in the prior artthat were based on Ad5, since those failed to provide completeprotection against RSV challenge replication after a singleintramuscular administration (Kim et al., 2010, Vaccine 28:3801-3808;Kohlmann et al., 2009, 1 Virol. 83:12601-12610; Krause et al., 2011,Virology Journal 8:375). The serotype further generally has a lowseroprevalence and/or low pre-existing neutralizing antibody titers inthe human population. Recombinant adenoviral vectors of this serotypeand of Ad35 with different transgenes are evaluated in clinical trials,and thus far show to have an excellent safety profile. Preparation ofrAd26 vectors is described, for example, in WO 2007/104792 and in Abbinket al. (2007) Virol. 81(9):4654-63. Exemplary genome sequences of Ad26are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO2007/104792. Preparation of rAd35 vectors is described, for example, inU.S. Pat. No. 7,270,811, in WO 00/70071, and in Vogels et al. (2003) J.Virol. 77(15):8263-71. Exemplary genome sequences of Ad35 are found inGenBank Accession AC_(—)000019 and in FIG. 6 of WO 00/70071.

A recombinant adenovirus hereof may be replication-competent orreplication-deficient.

In certain embodiments, the adenovirus is replication deficient, e.g.,because it contains a deletion in the E1 region of the genome. As knownto the skilled person, in case of deletions of essential regions fromthe adenovirus genome, the functions encoded by these regions have to beprovided in trans, preferably by the producer cell, i.e., when parts orwhole of E1, E2 and/or E4 regions are deleted from the adenovirus, thesehave to be present in the producer cell, for instance, integrated in thegenome thereof, or in the form of so-called helper adenovirus or helperplasmids. The adenovirus may also have a deletion in the E3 region,which is dispensable for replication, and hence such a deletion does nothave to be complemented.

A producer cell (sometimes also referred to in the art and herein as“packaging cell” or “complementing cell” or “host cell”) that can beused can be any producer cell wherein a desired adenovirus can bepropagated. For example, the propagation of recombinant adenovirusvectors is done in producer cells that complement deficiencies in theadenovirus. Such producer cells preferably have in their genome at leastan adenovirus E1 sequence, and thereby are capable of complementingrecombinant adenoviruses with a deletion in the El region. AnyE1-complementing producer cell can be used, such as human retina cellsimmortalized by E1, e.g., 911 or PER.C6® cells (see U.S. Pat. No.5,994,128), E1-transformed amniocytes (See EP Patent 1230354),E1-transformed A549 cells (see e.g., WO 98/39411, U.S. Pat. No.5,891,690), GH329:HeLa (Gao et al., 2000, Human Gene Therapy11:213-219), 293, and the like. In certain embodiments, the producercells are, for instance, HEK293 cells, or PER.C6® cells, or 911 cells,or IT293SF cells, and the like.

For non-subgroup C E1-deficient adenoviruses such as Ad35 (subgroup B)or Ad26 (subgroup D), it is preferred to exchange the E4-orf6 codingsequence of these non-subgroup C adenoviruses with the E4-orf6 of anadenovirus of subgroup C such as Ad5. This allows propagation of suchadenoviruses in well known complementing cell lines that express the E1genes of Ad5, such as, for example, 293 cells or PER.C6® cells (see,e.g., Havenga et al., 2006, J. Gen. Virol. 87:2135-2143; WO 03/104467,incorporated in its entirety by reference herein). In certainembodiments, an adenovirus that can be used is a human adenovirus ofserotype 35, with a deletion in the E1 region into which the nucleicacid molecule encoding RSV F protein antigen has been cloned, and withan E4 orf6 region of Ad5. In certain embodiments, the adenovirus in thevaccine composition hereof is a human adenovirus of serotype 26, with adeletion in the E1 region into which the nucleic acid molecule encodingRSV F protein antigen has been cloned, and with an E4 orf6 region ofAd5.

In alternative embodiments, there is no need to place a heterologousE4orf6 region (e.g., of Ad5) in the adenoviral vector, but instead theE1-deficient non-subgroup C vector is propagated in a cell line thatexpresses both E1 and a compatible E4orf6, e.g., the 293-ORF6 cell linethat expresses both E1 and E4orf6 from Ad5 (see e.g., Brough et al.,1996, 1 J. Virol. 70:6497-501 describing the generation of the 293-ORF6cells; Abrahamsen et al., 1997, 1 J. Virol. 71:8946-51 and Nan et al.,2003, Gene Therapy 10:326-36 each describing generation of E1-deletednon-subgroup C adenoviral vectors using such a cell line).

Alternatively, a complementing cell that expresses El from the serotypethat is to be propagated can be used (see e.g., WO 00/70071, WO02/40665).

For subgroup B adenoviruses, such as Ad35, having a deletion in the E1region, it is preferred to retain the 3′ end of the E1B 55K open readingframe in the adenovirus, for instance, the 166 by directly upstream ofthe pIX open reading frame or a fragment comprising this such as a 243by fragment directly upstream of the pIX start codon (marked at the 5′end by a Bsu36I restriction site in the Ad35 genome), since thisincreases the stability of the adenovirus because the promoter of thepIX gene is partly residing in this area (see, e.g., Havenga et al.,2006, 1 Gen. Virol. 87:2135-2143; WO 2004/001032, incorporated byreference herein).

“Heterologous nucleic acid molecule” (also referred to herein as“transgene”) in adenoviruses hereof is nucleic acid molecule that is notnaturally present in the adenovirus. It is introduced into theadenovirus, for instance, by standard molecular biology techniques. Inthis disclosure, the heterologous nucleic acid molecule encodes RSV Fprotein or fragment thereof. It can, for instance, be cloned into adeleted E1 or E3 region of an adenoviral vector. A transgene isgenerally operably linked to expression control sequences. This can, forinstance, be done by placing the nucleic acid molecule encoding thetransgene(s) under the control of a promoter. Further regulatorysequences may be added. Many promoters can be used for expression of atransgene(s), and are known to the skilled person. A non-limitingexample of a suitable promoter for obtaining expression in eukaryoticcells is a CMV-promoter (U.S. Pat. No. 5,385,839), e.g., the CMVimmediate early promoter, for instance, comprising nt. −735 to +95 fromthe CMV immediate early gene enhancer/promoter. A polyadenylationsignal, for example, the bovine growth hormone polyA signal (U.S. Pat.No. 5,122,458), may be present behind the transgene(s).

In certain embodiments, the recombinant adenovirus vectors comprise asthe 5′ terminal nucleotides the nucleotide sequence: CTATCTAT. Theseembodiments are advantageous because such vectors display improvedreplication in production processes, resulting in batches of adenoviruswith improved homogeneity, as compared to vectors having the original 5′terminal sequences (generally CATCATCA) (see also Patent ApplicationNos. PCT/EP2013/054846 and U.S. Pat. No. 13/794,318, entitled “Batchesof recombinant adenovirus with altered terminal ends” filed on Mar. 12,2012 in the name of Crucell Holland B.V.), incorporated in its entiretyby reference herein. Thus, also provided are batches of recombinantadenovirus encoding RSV F protein or a part thereof, wherein theadenovirus is a human adenovirus serotype 26, and wherein essentiallyall (e.g., at least 90%) of the adenoviruses in the batch comprise agenome with terminal nucleotide sequence CTATCTAT.

The F protein of RSV may be derived from any strains of naturallyoccurring or recombinant RSV, preferably from human RSV strains, such asA2, Long, or B strains. In further embodiments, the sequence may be aconsensus sequence based upon a plurality of RSV F protein amino acidsequences. In one example hereof, the RSV strain is RSV-A2 strain.

The F protein of RSV may be the full length of F protein of RSV, orfragment thereof. In one embodiment, the nucleotide sequence encoding Fprotein of RSV encodes the full length of F protein of RSV (F0), such asthe amino acid of SEQ ID NO:1. In one example, the nucleotide sequenceencoding F protein of RSV has the nucleotide sequence of SEQ ID NO:2.Alternatively, the sequence encoding F protein of RSV may be anysequence that is at least about 80%, preferably more than about 90%,more preferably at least about 95%, identical to the nucleotide sequenceof SEQ ID NO:2. In other embodiments, codon-optimized sequences such as,for instance, provided in SEQ ID NO:2, 4, 5 or 6 of WO 2012/021730 canbe used.

In another embodiment, the nucleotide sequence may alternatively encodea fragment of F protein of RSV. The fragment may result from either orboth of amino-terminal and carboxy-terminal deletions. The extent ofdeletion may be determined by a person skilled in the art to, forexample, achieve better yield of the recombinant adenovirus. Thefragment will be chosen to comprise an immunologically active fragmentof the F protein, i.e., a part that will give rise to an immune responsein a subject. This can be easily determined using in silico, in vitroand/or in vivo methods, all routine to the skilled person. In oneembodiment of this disclosure, the fragment is a transmembrane codingregion-truncated F protein of RSV (F0ΔTM, see e.g., U.S. Patent20110014220). The fragments of F protein may also be F1 domain or F2domain of F protein. The fragments of F may also be fragments containingneutralization epitopes and T cell epitopes (Sing et al., 2007, Virol.Immunol. 20, 261-275; Sing et al., 2007, Vaccine 25, 6211-6223).

The term “about” for numerical values as used in the present disclosuremeans the value ±10%.

In certain embodiments, provided are methods for making a vaccineagainst respiratory syncytial virus (RSV), comprising providing arecombinant human adenovirus of serotype 26 that comprises nucleic acidmolecule encoding a RSV F protein or fragment thereof, propagating saidrecombinant adenovirus in a culture of host cells, isolating andpurifying the recombinant adenovirus, and bringing the recombinantadenovirus in a pharmaceutically acceptable composition.

Recombinant adenovirus can be prepared and propagated in host cells,according to well known methods, which entail cell culture of the hostcells that are infected with the adenovirus. The cell culture can be anytype of cell culture, including adherent cell culture, e.g., cellsattached to the surface of a culture vessel or to microcarriers, as wellas suspension culture.

Most large-scale suspension cultures are operated as batch or fed-batchprocesses because they are the most straightforward to operate and scaleup. Nowadays, continuous processes based on perfusion principles arebecoming more common and are also suitable (see e.g., WO 2010/060719,and WO 2011/098592, both incorporated by reference herein, whichdescribe suitable methods for obtaining and purifying large amounts ofrecombinant adenoviruses).

Producer cells are cultured to increase cell and virus numbers and/orvirus titers. Culturing a cell is done to enable it to metabolize,and/or grow and/or divide and/or produce virus of interest hereof. Thiscan be accomplished by methods as such well known to persons skilled inthe art, and includes but is not limited to providing nutrients for thecell, for instance, in the appropriate culture media. Suitable culturemedia are well known to the skilled person and can generally be obtainedfrom commercial sources in large quantities, or custom-made according tostandard protocols. Culturing can be done, for instance, in dishes,roller bottles or in bioreactors, using batch, fed-batch, continuoussystems and the like. Suitable conditions for culturing cells are known(see e.g., Tissue Culture, Academic Press, Kruse and Paterson, editors(1973), and R. I. Freshney, Culture of Animal Cells: A Manual of BasicTechnique, fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9).

Typically, the adenovirus will be exposed to the appropriate producercell in a culture, permitting uptake of the virus. Usually, the optimalagitation is between about 50 and 300 rpm, typically about 100-200,e.g., about 150, typical DO is 20-60%, e.g., 40%, the optimal pH isbetween 6.7 and 7.7, the optimal temperature between 30 and 39° C.,e.g., 34-37° C., and the optimal MOI between 5 and 1000, e.g., about50-300. Typically, adenovirus infects producer cells spontaneously, andbringing the producer cells into contact with rAd particles issufficient for infection of the cells. Generally, an adenovirus seedstock is added to the culture to initiate infection, and subsequentlythe adenovirus propagates in the producer cells. This is all routine forthe person skilled in the art.

After infection of an adenovirus, the virus replicates inside the celland is thereby amplified, a process referred to herein as propagation ofadenovirus. Adenovirus infection results finally in the lysis of thecells being infected. The lytic characteristics of adenovirus thereforepermits two different modes of virus production. The first mode isharvesting virus prior to cell lysis, employing external factors to lysethe cells. The second mode is harvesting virus supernatant after(almost) complete cell lysis by the produced virus (see e.g., U.S. Pat.No. 6,485,958, describing the harvesting of adenovirus without lysis ofthe host cells by an external factor). It is preferred to employexternal factors to actively lyse the cells for harvesting theadenovirus.

Methods that can be used for active cell lysis are known to the personskilled in the art, and have, for instance, been discussed in WO98/22588, p. 28-35. Useful methods in this respect are, for example,freeze-thaw, solid shear, hypertonic and/or hypotonic lysis, liquidshear, sonication, high pressure extrusion, detergent lysis,combinations of the above, and the like. In one embodiment, the cellsare lysed using at least one detergent. Use of a detergent for lysis hasthe advantage that it is an easy method, and that it is easily scalable.

Detergents that can be used, and the way they are employed, aregenerally known to the person skilled in the art. Several examples are,for instance, discussed in WO 98/22588, p. 29-33. Detergents can includeanionic, cationic, zwitterionic, and nonionic detergents. Theconcentration of the detergent may be varied, for instance, within therange of about 0.1%-5% (w/w). In one embodiment, the detergent used isTRITON® X-100.

Nuclease may be employed to remove contaminating, i.e., mostly from theproducer cell, nucleic acid molecules. Exemplary nucleases suitable foruse in this disclosure include BENZONASE®, PULMOZYME®, or any otherDNase and/or RNase commonly used within the art. In preferredembodiments, the nuclease is BENZONASE®, which rapidly hydrolyzesnucleic acid molecules by hydrolyzing internal phosphodiester bondsbetween specific nucleotides, thereby reducing the viscosity of the celllysate. BENZONASE® can be commercially obtained from Merck KGaA (codeW214950). The concentration in which the nuclease is employed ispreferably within the range of 1-100 units/ml. Alternatively, or inaddition to nuclease treatment, it is also possible to selectivelyprecipitate host cell DNA away from adenovirus preparations duringadenovirus purification, using selective precipitating agents such asdomiphen bromide (see e.g., U.S. Pat. No. 7,326,555; Goerke et al.,2005, Biotechnology and Bioengineering, Vol. 91:12-21; WO 2011/045378;WO 2011/045381).

Methods for harvesting adenovirus from cultures of producer cells havebeen extensively described in WO 2005/080556.

In certain embodiments, the harvested adenovirus is further purified.Purification of the adenovirus can be performed in several stepscomprising clarification, ultrafiltration, diafiltration or separationwith chromatography as described in, for instance, WO 05/080556,incorporated by reference herein. Clarification may be done by afiltration step, removing cell debris and other impurities from the celllysate. Ultrafiltration is used to concentrate the virus solution.Diafiltration, or buffer exchange, using ultrafilters is a way forremoval and exchange of salts, sugars and the like. The person skilledin the art knows how to find the optimal conditions for eachpurification step. Also WO 98/22588, incorporated in its entirety byreference herein, describes methods for the production and purificationof adenoviral vectors. The methods comprise growing host cells,infecting the host cells with adenovirus, harvesting and lysing the hostcells, concentrating the crude lysate, exchanging the buffer of thecrude lysate, treating the lysate with nuclease, and further purifyingthe virus using chromatography.

Preferably, purification employs at least one chromatography step, as,for instance, discussed in WO 98/22588, p. 61-70. Many processes havebeen described for the further purification of adenoviruses, whereinchromatography steps are included in the process. The person skilled inthe art will be aware of these processes, and can vary the exact way ofemploying chromatographic steps to optimize the process. It is, forinstance, possible to purify adenoviruses by anion exchangechromatography steps, see, for instance, WO 2005/080556 and Konz et al.,2005, Hum. Gene Ther. 16:1346-1353. Many other adenovirus purificationmethods have been described and are within the reach of the skilledperson. Further methods for producing and purifying adenoviruses aredisclosed in, for example (WO 00/32754; WO 04/020971; U.S. Pat. No.5,837,520; U.S. Pat. No. 6,261,823; WO 2006/108707; Konz et al., 2008,Methods Mol. Biol. 434:13-23; Altaras et al., 2005, Adv. Biochem. Eng.Biotechnol. 99:193-260), all incorporated by reference herein.

For administering to humans, the invention may employ pharmaceuticalcompositions comprising the rAd and a pharmaceutically acceptablecarrier or excipient. In the present context, the term “Pharmaceuticallyacceptable” means that the carrier or excipient, at the dosages andconcentrations employed, will not cause any unwanted or harmful effectsin the subjects to which they are administered. Such pharmaceuticallyacceptable carriers and excipients are well known in the art (see,Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,Mack Publishing Company (1990); Pharmaceutical Formulation Developmentof Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press (2000)). The purified rAd preferablyis formulated and administered as a sterile solution although it is alsopossible to utilize lyophilized preparations. Sterile solutions areprepared by sterile filtration or by other methods known per se in theart. The solutions are then lyophilized or filled into pharmaceuticaldosage containers. The pH of the solution generally is in the range ofpH 3.0 to 9.5, e.g., pH 5.0 to 7.5. The rAd typically is in a solutionhaving a suitable pharmaceutically acceptable buffer, and the solutionof rAd may also contain a salt. Optionally stabilizing agent may bepresent, such as albumin. In certain embodiments, detergent is added. Incertain embodiments, rAd may be formulated into an injectablepreparation. These formulations contain effective amounts of rAd, areeither sterile liquid solutions, liquid suspensions or lyophilizedversions and optionally contain stabilizers or excipients. An adenovirusvaccine can also be aerosolized for intranasal administration (see e.g.,WO 2009/117134).

For instance, adenovirus may be stored in the buffer that is also usedfor the Adenovirus World Standard (Hoganson et al., Development of astable adenoviral vector formulation, Bioprocessing March 2002, p.43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol. Another usefulformulation buffer suitable for administration to humans is 20 mM Tris,2 mM MgCl₂, 25 mM NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v.Obviously, many other buffers can be used, and several examples ofsuitable formulations for the storage and for pharmaceuticaladministration of purified (adeno)virus preparations can, for instance,be found in European Patent 0853660, U.S. Pat. No. 6,225,289 and inInternational Patent Applications WO 99/41416, WO 99/12568, WO 00/29024,WO 01/66137, WO 03/049763, WO 03/078592, and WO 03/061708.

In certain embodiments, a composition comprising the adenovirus furthercomprises one or more adjuvants. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant, and pharmaceutical compositions comprising adenovirus andsuitable adjuvants are, for instance, disclosed in WO 2007/110409,incorporated by reference herein. The terms “adjuvant” and “immunestimulant” are used interchangeably herein, and are defined as one ormore substances that cause stimulation of the immune system. In thiscontext, an adjuvant is used to enhance an immune response to theadenovirus vectors hereof Examples of suitable adjuvants includealuminum salts such as aluminum hydroxide and/or aluminum phosphate;oil-emulsion compositions (or oil-in-water compositions), includingsqualene-water emulsions, such as MF59 (see e.g., WO 90/14837); saponinformulations, such as, for example, QS21 and Immunostimulating Complexes(ISCOMS) (see e.g., U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711,WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives,examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL(3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylatingbacterial toxins or mutants thereof, such as E. coli heat labileenterotoxin LT, cholera toxin CT, and the like. It is also possible touse vector-encoded adjuvant, e.g., by using heterologous nucleic acidmolecule that encodes a fusion of the oligomerization domain ofC4-binding protein (C4bp) to the antigen of interest (e.g., Solabomi etal., 2008, Infect. Immun. 76:3817-23). In certain embodiments, thecompositions hereof comprise aluminum as an adjuvant, e.g., in the formof aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate,or combinations thereof, in concentrations of 0.05-5 mg, e.g., from0.075-1.0 mg, of aluminum content per dose.

In other embodiments, the compositions do not comprise adjuvants.

It is also possible hereof to administer further active components, incombination with the vaccines hereof Such further active components maycomprise, e.g., other RSV antigens or vectors comprising nucleic acidmolecule encoding these. Such vectors may be non-adenoviral oradenoviral, of which the latter can be of any serotype. An example ofother RSV antigens includes RSV G protein or immunologically activeparts thereof For instance, intranasally applied recombinantreplication-deficient Ad5-based adenovector rAd/3×G, expressing thesoluble core domain of G glycoprotein (amino acids 130 to 230) wasprotective in a murine model (Yu et al., 2008, J. Virol. 82:2350-2357),and although it was not protective when applied intramuscularly, it isclear from these data that RSV G is a suitable antigen for inducingprotective responses. Further active components may also comprisenon-RSV antigens, e.g., from other pathogens such as viruses, bacteria,parasites, and the like. The administration of further active componentsmay, for instance, be done by separate administration or byadministering combination products of the vaccines hereof and thefurther active components. In certain embodiments, furthernon-adenoviral antigens (besides RSV.F), may be encoded in the vectors.In certain embodiments, it may, thus, be desired to express more thanone protein from a single adenovirus, and in such cases more codingsequences, for instance, may be linked to form a single transcript froma single expression cassette or may be present in two separateexpression cassettes cloned in different parts of the adenoviral genome.

Adenovirus compositions may be administered to a subject, e.g., a humansubject. The total dose of the adenovirus provided to a subject duringone administration can be varied as is known to the skilledpractitioner, and is generally between 1×10⁷ viral particles (vp) and1×10¹² vp, preferably between 1×10⁸ vp and 1×10¹¹ vp, for instance,between 3×10⁸ and 5×10¹ vp, for instance, between 10⁹ and 3×10¹ vp.

Administration of adenovirus compositions can be performed usingstandard routes of administration. Non-limiting embodiments includeparenteral administration, such as by injection e.g., intradermal,intramuscular, etc., or subcutaneous, transcutaneous, or mucosaladministration, e.g., intranasal, oral, and the like. Intranasaladministration has generally been seen as a preferred route for vaccinesagainst RSV. The most important advantage of the live intrasal strategyis the direct stimulation of local respiratory tract immunity and thelack of associated disease enhancement. The only vaccines under clinicalevaluation for pediatric use at the present time are live intranasalvaccine (Collins and Murphy, Vaccines against human respiratorysyncytial virus, in Perspectives in Medical Virology 14: RespiratorySyncytial Virus (Ed. P. Cane), Elsevier, Amsterdam, the Netherlands, pp.233-277). Intranasal administration is a suitable preferred routeaccording to this disclosure as well. However, it is particularlypreferred according to this disclosure to administer the vaccineintramuscularly, since it was surprisingly found that intramuscularadministration of the vaccine hereof resulted in protection against RSVreplication in nose and lungs of cotton rats, unlike earlier reportedintramuscular RSV vaccines based on other adenovirus serotypes. Theadvantage of intramuscular administration is that it is simple andwell-established, and does not carry the safety concerns for intranasalapplication in infants younger than 6 months. In one embodiment acomposition is administered by intramuscular injection, e.g., into thedeltoid muscle of the arm, or vastus lateralis muscle of the thigh. Theskilled person knows the various possibilities to administer acomposition, e.g., a vaccine in order to induce an immune response tothe antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance, a rodent,e.g., a mouse, a cotton rat, or a non-human-primate, or a human.Preferably, the subject is a human subject. The subject can be of anyage, e.g., from about 1 month to 100 years old, e.g., from about 2months to about 80 years old, e.g., from about 1 month to about 3 yearsold, from about 3 years to about 50 years old, from about 50 years toabout 75 years old, etc.

It is also possible to provide one or more booster administrations ofone or more adenovirus vaccines hereof. If a boosting vaccination isperformed, typically, such a boosting vaccination will be administeredto the same subject at a moment between one week and one year,preferably between two weeks and four months, after administering thecomposition to the subject for the first time (which is in such casesreferred to as “priming vaccination”). In alternative boosting regimens,it is also possible to administer different vectors, e.g., one or moreadenoviruses of different serotype, or other vectors such as MVA, orDNA, or protein, to the subject after the priming vaccination. It is,for instance, possible to administer to the subject a recombinantadenoviral vector hereof as a prime, and boosting with a compositioncomprising RSV F protein.

In certain embodiments, the administration comprises a priming and atleast one booster administration. In certain embodiments thereof, thepriming administration is with a rAd35 comprising nucleic acid moleculeencoding RSV F protein or a fragment thereof (“rAd35-RSV.F”) and thebooster administration is with a rAd26 comprising nucleic acid moleculeencoding RSV F protein hereof (“rAd26-RSV.F”). In other embodimentsthereof, the priming administration is with rAd26-RSV.F and the boosteradministration is with rAd35-RSV.F. In other embodiments, both thepriming and booster administration are with rAd26.RSV.F. In certainembodiments, the priming administration is with rAd26-RSV.F and thebooster administration is with RSV F protein. In all these embodiments,it is possible to provide further booster administrations with the sameor other vectors or protein. Embodiments where boosting with RSV Fprotein may be particularly beneficial include e.g., in elder subjectsin risk groups (e.g., having COPD or asthma) of 50 years or older, ore.g., in healthy subjects of 60 years or older or 65 years or older.

In certain embodiments, the administration comprises a singleadministration of a recombinant adenovirus hereof, without further(booster) administrations. Such embodiments are advantageous in view ofthe reduced complexity and costs of a single administration regimen ascompared to a prime-boost regimen. Complete protection is alreadyobserved after single administration of the recombinant adenoviralvectors hereof without booster administrations in the cotton rat modelin the examples herein.

The invention is further explained in the following examples. Theexamples do not limit the invention in any way. They merely serve toclarify the invention.

EXAMPLES Example 1

Preparation of Adenoviral Vectors

Cloning RSV F gene into El region of Ad35 and Ad26:

The RSV.F(A2)nat gene, coding for the native RSV fusion (F) protein ofthe A2 strain (Genbank AC083301.1), was gene optimized for humanexpression and synthesized, by Geneart. A Kozak sequence (5′ GCCACC 3′)was included directly in front of the ATG start codon, and two stopcodons (5′ TGA TAA 3′) were added at the end of the RSV.F(A2)nat codingsequence. The RSV.F(A2)nat gene was inserted in the pAdApt35BSU plasmidand in the pAdApt26 plasmid via HindIII and XbaI sites. The resultingplasmids, pAdApt35BSU.RSV.F(A2)nat and pAdApt26.RSV.F(A2)nat aredepicted in FIG. 15. The amino acid sequence of the F protein and thecodon optimized sequence encoding that amino acid sequence are providedherein as SEQ ID NOs:1 and 2 (SEQ ID NO:1: RSV fusion protein (GenbankACO83301.1) amino acid sequence and SEQ ID NO:2: codon optimizedRSV.F(A2)nat gene that codes for the RSV fusion protein), respectively.

Cell culture:

PER.C6® cells (Fallaux et al., 1998, Hum. Gene. Ther. 9:1909-1917) weremaintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetalbovine serum (FBS), supplemented with 10 mM MgCl₂.

Adenovirus generation, infections and passaging:

All adenoviruses were generated in PER.C6® cells by single homologousrecombination and produced as previously described (for rAd35: Havengaet al., 2006, J. Gen. Virol. 87:2135-2143; for rAd26: Abbink et al.,2007,1 Virol. 81:4654-4663). Briefly, PER.C6® cells were transfectedwith Ad vector plasmids, using Lipofectamine according to theinstructions provided by the manufacturer (Life Technologies). Forrescue of Ad35 vectors carrying the RSV.F(A2)nat transgene expressioncassette, the pAdApt35BSU.RSV.F(A2)nat plasmid andpWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used, whereas for Ad26 vectorscarrying the RSV.F(A2)nat transgene expression cassette, thepAdApt26.RSV.F(A2)nat plasmid and pWE.Ad26.dE3.5orf6.cosmid were used.Cells were harvested one day after full CPE, freeze-thawed, centrifugedfor 5 minutes at 3,000 rpm, and stored at −20° C. Next the viruses wereplaque purified and amplified in PER.C6® cultured on a single well of amultiwell 24 tissue culture plate. Further amplification was carried outin PER.C6® cultured using a T25 tissue culture flask and a T175 tissueculture flask. Of the T175 crude lysate, 3 to 5 ml was used to inoculate20× T175 triple-layer tissue culture flasks containing 70% confluentlayers of PER.C6® cells. The virus was purified using a two-step CsC1purification method. Finally, the virus was stored in aliquots at −85°C.

Example 2

Induction of immunity against RSV F using recombinant adenovirusserotypes 26 and 35 in vivo.

This is an experiment to investigate the ability of the recombinantadenovirus serotype (Ad26) and recombinant adenovirus serotype 35 (Ad35)to induce immunity against the glycoprotein F antigen of RSV in BALB/cmice.

In this study animals were distributed in experimental groups of fivemice. Animals were immunized with a single dose of Ad26 or Ad35 carryingthe full-length RSV F gene (Ad26-RSV.F or Ad35-RSV.F) or no transgene(Ad26e or Ad35e). Three ten-fold serial dilutions of rAd ranging from10¹⁰ to 10⁸ virus particles (vp) were given intramuscularly. Ascontrols, one group of three animals received the empty vector Ad26e andone group received the empty vector Ad35e.

The ELISPOT assay is used to determine the relative number of Fprotein-specific IFNγ-secreting T cells in the spleen, and isessentially done as described by Rado{hacek over (s)}vić et al. (Clin.Vaccine Immunol. 2010; 17(11):1687-94.). For the stimulation ofsplenocytes in the ELISPOT assay, two peptide pools consisting of11-amino-acid-overlapping 15-mer peptides spanning the whole sequence ofthe RSV F (A2) protein was used. The numbers of spot-faulting units(SFU) per 10⁶ cells were calculated.

For the determination of antibody titers an ELISA assay was used. Forthis, ELISA plates (Thermo Scientific) were coated with 25 μg/ml RSVLong whole inactivated antigen (Virion Serion, cat# BA113VS). Dilutedserum samples were added to the plates, and IgG antibodies against RSVwere determined using biotin-labeled anti-Mouse IgG (DAKO, cat# E0413),using detection by horseradish peroxidase (PO)-conjugated streptavidin(SA). Titers were calculated by linear interpolation, using 1.5× ODsignal from 50× diluted naïve serum as cut-off The titers ofRSV-Specific IgG1 and IgG2a antibodies in the serum of the mouse wasdetermined using PO-labeled anti-mouse IgG1 and PO-labeled anti-mouseIgG2a (Southern Biotechnology Associates, cat. #s 1070-05 and 1080-05)were used to quantify subclasses.

Virus neutralizing activity (VNA) of the antibodies was determined bymicroneutralization assay, essentially done as described by Johnson etal. (J. Infect. Dis. 1999 Jul; 180(1):35-40). RSV-susceptible VERO cellswere seeded in 96-well cell-culture plates one day prior to infection.On the day of infection, serial diluted sera and controls were mixedwith 1200 pfu of RSV (Long or B1) and incubated 1 hour at 37° C.Subsequently, virus/antibody mixes were transferred to 96-wells platescontaining VERO cell monolayers. Three days later monolayers were fixedwith 80% ice-cold acetone and RSV antigen was determined with an anti-Fmonoclonal antibody. The neutralizing titer is expressed as the serumdilution (log₂) that causes 50% reduction in the OD450 from virus-onlycontrol wells (IC₅₀).

At week 2 and week 8 post-prime, animals were sacrificed and cellularand humoral responses were monitored as described above.

FIG. 1 shows that all doses of Ad26-RSV.F (FIG. 1A) and Ad35-RSV.F (FIG.1B) were effective in inducing good cellular immune responses and thatthe responses were stable over time. No significant differences ofvector dose on T cell response with either Ad26-RSV.F or Ad35-RSV.F wereobserved.

FIG. 2 shows the antibody titers in the same experiment as describedabove. Both vectors induced very clear time and dose-dependent increasein ELISA titers (FIG. 2). Anti-F titers clearly increase from 2 to 8weeks, which was significant for the 10¹⁰ dose. At 8 weeks there was nodifference in titers between the Ad26-RSV.F or Ad35-RSV.F vectors.

The subclass distribution (IgG1 vs IgG2a) of F-specific IgG wasdetermined to evaluate the balance of Th1 vs Th2 response. A skewedTh2/Th1 response predispose animals to develop vaccine-enhanced RSVdisease as seen with formalin-inactivated RSV. As shown in FIG. 3, theIgG2a/IgG1 ratio for both Ad26-RSV.F and Ad35-RSV.F is higher than 1.This strongly indicates that adenovectors Ad26-RSV.F and Ad35-RSV.Fexhibit rather a Th1 type than a Th2 type of response.

FIG. 4 shows the virus neutralizing titers (VNA) of the same sera usedfor the antibody titers. Immunization with Ad26-RSV.F and rAd35-RSV.Fled to the induction of neutralizing antibody titers. VNA titersstrongly increased between two and eight weeks post-prime in mice given10¹⁰ vp. At eight weeks there was no difference in titers betweenAd26-RSV.F and Ad35-RSV.F vectors in mice given 10¹⁰ vp.

From these immunization experiments it is evident that Ad35 and Ad26vectors harboring the RSV.F transgene induce strong cellular and humoralresponses against RSV.F.

Example 3

Immunity against RSV.F after heterologous prime-boost using recombinantadenoviral vectors encoding RSV.F.

This study was designed to investigate the ability of prime-boostregimens based on adenoviral vectors derived from two differentserotypes to induce immunity against RSV.F.

This study involved BALB/c mice distributed in experimental groups ofeight mice. Animals were immunized by intramuscular injection with 10¹⁰vp carrying the wild-type sequence of the RSV.F gene based on/derivedfrom RSV A2 (Ad-RSV.F or Ad35-RSV.F) or no transgene (Ad26e or Ad35e).One group of animals was primed with Ad26-RSV.F and boosted at week 4with Ad35-RSV.F or Ad35e. Another group of animals was primed withAd35-RSV.F and boosted at week 4 with Ad26-RSV.F or Ad26e. A controlgroup of mice was primed with Ad35e and boosted at week 4 with Ad26e. Atweek 6 and week 12 post prime, eight animals were sacrificed at eachtime point and cellular and humoral responses were monitored withimmunological assays well known to persons skilled in the art and asdescribed above.

FIG. 5 shows the cellular response at 6 and 12 weeks after the firstimmunization. At 6 weeks after prime (and 2 weeks post-boost), asignificant boost effect by both Ad26-RSV.F and Ad35-RSV.F on T cellresponses was measured, and the magnitude of T cell response wasindependent of order of immunization with Ad26-RSV.F or Ad35-RSV.F inprime-boost. At 12 weeks after prime (8 weeks post-boost), mice primedwith Ad26-RSV.F had maintained higher levels of F-specific T cellseither in primed-only and prime-boosted animals, compared to rAd35-RSV.Fprimed animals. Overall, the numbers of F-specific lymphocytes (SFU)were high and stable for at least 12 weeks in all animals immunized witheither rAd26-RSV.F or rAd35-RSV.F (prime/or prime-boost).

FIG. 6 shows the humoral response at different time points afterprime-boost vaccination with the adenoviral vectors. Ad35.RSV.F andAd26.RSV.F prime equally well, and a significant boost effect induced byeither Ad26.RSV.F or rAd35.RSV.F on B cell responses was shown.Moreover, the magnitude of B cell responses in heterologous prime-boostwas independent of the order of Ad35.RSV.F and Ad26.RSV.F immunization,and after boost ELISA titers remained stable for 12 weeks.

FIG. 7 shows the virus neutralizing antibody titers at different timepoints after prime-boost immunization. Both Ad35.RSV.F and Ad26.RSV.Fvectors primed equally well to achieve clear VNA titers, as was observedfor ELISA titers. Also, the increase in VNA titers after heterologousprime-boost was independent of the order of Ad35.RSV.F and Ad26.RSV.Fimmunization. Boost effect by either Ad26.RSV.F or Ad35.RSV.F on VNAtiters was significant at both time-points and already maximal at 6weeks. Groups that were only primed with Ad.RSV.F have increased VNAtiters at 12 weeks compared to 6 weeks. The RSV F sequence in theadenoviral vector constructs is derived from the RSV A2 isolate. Theneutralizing assay described in this application is based on RSV Longstrain, belonging to RSV subgroup A, demonstrating that the antibodiesinduced by F (A2) are able to cross-neutralize a different RSV A strainsubtype.

Because the RSV F protein is well conserved among RSV isolates, it wastested whether sera from animals immunized with Ad-RSV.F vectors wereable to cross-neutralize a prototypical RSV B strain isolate, RSV B 1.As shown in FIG. 8, sera of immunized mice were also capable ofcross-neutralizing the B1 strain. The capacity to cross-neutralize RSVB1 was not dependent on which vector was used in prime-only groups, ororder of prime-boost immunization with Ad26.RSV.F and Ad35.RSV.Fvectors.

Collectively, these data show that in a prime-boost regimen, consecutiveimmunizations with Ad26.RSV.F and Ad35.RSV.F induce strong humoral andcellular responses, and that the humoral immune response includes thecapacity to neutralize isolates of both RSV A and B subtypes.

Example 4

Inducing protection against RSV infection using recombinant adenoviralvectors in vivo in a cotton rat model.

This experiment was performed to investigate the ability of prime-boostregimens based on adenoviral vectors derived from two differentserotypes to induce protection against RSV challenge replication in thecotton rat. Cotton rats (Sigmodon hispidus) are susceptible to bothupper and lower respiratory tract infection with RSV and were found tobe at least 50-fold more permissive than mouse strains (Niewiesk et al.,2002, Lab. Anim. 36(4):357-72). Moreover the cotton rat has been theprimary model assessing the efficacy and safety of RSV candidatevaccines, antivirals and antibodies. Preclinical data generated in thecotton rat model advanced the development of two antibody formulations(RESPIGAM® and SYNAGIS®) to clinical trials without the need ofintermediate studies in non-human primates.

The study enrolled cotton rats in experimental groups of eight cottonrats each. Animals were immunized by intramuscular injections of 10⁹viral particles (vp) or 10¹⁰ vp adenoviral vectors carrying thefull-length RSV F (A2) gene (Ad26.RSV.F or Ad35.RSV.F) or no transgene(Ad26e or Ad35e). Animals were boosted 28 days later with the same vpdose, either with the same vector (homologous prime-boost) or with otheradenoviral serotype (heterologous prime-boost); control groups wereimmunized accordingly with Ad-e vectors, except that only one dose wasapplied (10¹⁰). Control groups consisted of six animals. Animalsinfected intranasally with RSV A2 (10⁴ plaque forming units (pfu)) wereused as positive control for protection against challenge replication,as it is known that primary infection with RSV virus protects againstsecondary challenge replication (Prince, Lab. Invest. 1999,79:1385-1392). Furthermore, formalin-inactivated RSV (FI-RSV) served ascontrol for vaccine-enhanced histopathological disease. Three weeksafter the second (boost) immunization, the cotton rats were challengedintranasally with 1×10⁵ pfu of plaque-purified RSV A2. As controls, onegroup of cotton rats was not immunized but received challenge virus, andanother control group was not immunized and not challenged. Cotton ratswere sacrificed 5 days after infection, a time point at which RSVchallenge virus reaches peak titers (Prince, Lab. Invest. 1999,79:1385-1392), and lung and nose RSV titers were determined by virusplaque titration (Prince et al., 1978, Am. J. Pathology 93:711-791).

FIG. 9 shows that high RSV virus titers in lungs and in nose wereobserved in non-immunized controls as well as animals receivingadenoviral vectors without transgene, respectively 5.3+/−0.13 log₁₀pfu/gram and 5.4+/−0.35 log₁₀ pfu. In contrast, no challenge virus couldbe detected in lung and nose tissue from animals that receivedprime-boost immunization with Ad26.RSV.F and/or Ad35.RSV.F vectors,independent of dose or regimen.

These data clearly demonstrate that both Ad35-based and Ad26-basedvectors give complete protection against RSV challenge replication inthe cotton rat model. This was surprising, as Ad5-based adenoviralvectors encoding RSV F were known not to be capable of inducing completeprotection in animal models after intramuscular administration.

In the course of the experiment, blood samples were taken beforeimmunization (day 0), before the boost immunization (day 28), at day ofchallenge (day 49) and at day of sacrifice (day 54). The sera weretested in a plaque assay-based virus neutralization assay (VNA) for theinduction of systemic RSV specific neutralizing antibodies as describedby Prince (Prince et al., 1978, Am. J. Pathology 93:711-791). Theneutralizing titer is expressed as the serum dilution (log₂) that causes50% plaque reduction compared to from virus-only control wells (IC₅₀).

FIG. 10 shows that control animals do not have virus neutralizingantibodies at day 28 and day 49, while high VNA titers are induced afteranimals were primed with Ad26.RSV.F or Ad35.RSV.F vectors. A moderateincrease in VNA titer is observed after boost immunizations. Primaryinfection with RSV A2 virus resulted in rather moderate VNA titers thatgradually increased in time.

To evaluate whether Ad26.RSV.F or Ad35.RSV.F vaccine might exacerbatedisease following a challenge with RSV A2, histopathological analyses ofthe lungs were performed 5 days after infection. The lungs wereharvested, perfused with formalin, sectioned, and stained withhematoxylin and eosin for histologic examination. Histopathology scorewas done blinded, according to criteria published by Prince (Prince etal., Lab. Invest. 1999, 79:1385-1392), and scored for the followingparameters: peribronchiolitis, perivasculitis, interstitial pneumonitis,and alveolitis. FIG. 11 shows the scoring of lung pathology of thisexperiment. Following RSV challenge, FI-RSV immunized animals showedelevated histopathology on all histopathology parameters examined,compared to mock-immunized challenged animals, which was expected basedon earlier published studies (Prince et al., Lab. Invest. 1999,79:1385-1392). Histopathology scores in Ad26.RSV.F and Ad35.RSV.Fimmunized compared to rAd-e or mock immunized animals, were similar,although perivasculitis in the rAd-RSV.F immunized animals appeared tobe slightly lower. Thus, the Ad26.RSV.F and Ad35.RSV.F vaccines did notresult in enhanced disease, unlike FI-RSV vaccines.

All vaccination strategies resulted in complete protection against RSVchallenge replication, induced strong virus neutralizing antibodies, andenhanced pathology was not observed.

Example 5

Protective efficacy of rAd vectors using different administration routesafter single immunization.

This study is to investigate the influence of administration routes onthe protective efficacy induced by Ad26 or Ad35 vectors encoding RSV.F.The vaccine was either administered intramuscularly or intranasally.

Cotton rats that had received a single immunization with 1×10⁹ or 1×10¹viral particles (vp) of Ad26 or Ad35 carrying either the RSV F astransgene (Ad26.RSV.F or Ad35.RSV.F) or no transgene (Ad26-e or Ad35-e)at day 0, were challenged at day 49 with 10⁵ RSV pfu and sacrificed atday 54.

FIG. 12 shows the results of the experiments wherein the lung and nasalchallenge virus were determined. High RSV virus titers were detected inlungs and noses from rats that were non-immunized or immunized withadenoviral vectors without a transgene, respectively 4.9+/−0.22 log₁₀pfu/gram and 5.4+/−0.16 log₁₀ pfu. In contrast, lungs and noses fromanimals that received either Ad35-RSV.F or Ad26-RSV.F were devoid ofreplicating challenge virus, independent of administration route anddose.

These data surprisingly demonstrate that each of Ad26- and Ad35-basedvectors encoding RSV F protein provide complete protection in cotton ratchallenge experiments, independent of the route of administration of thevectors. This was unexpected, since none of the publishedadenovirus-based RSV vaccines, which were based on other serotypes, haddemonstrated complete protection after intramuscular vaccination.

During the experiment, blood samples were taken before immunization (day0), 4 weeks after immunization (day 28), and at day of challenge (day49). The sera were tested in a neutralization test for the induction ofRSV specific antibodies (FIG. 13). Prior to immunization no virusneutralizing antibodies were detected in any cotton rat. All adenoviralvector immunization strategies, independent of route of administration,clearly induced high VNA titers, which remained stable over time. Thesedata surprisingly demonstrate that each of Ad26-and Ad35-based vectorsencoding RSV F protein provide high titers of virus neutralizingantibodies in cotton rat immunization experiments, independent of theroute of administration of the vectors.

To evaluate whether a single immunization of Ad26.RSV.F or Ad35.RSV.Fvaccine can cause vaccine-enhanced disease following challenge with RSVA2, histopathological analyses of the lungs were performed 5 days afterinfection (FIG. 14). Single immunization with rAd26.RSV.F or rAd35.RSV.Fresulted in similar immunopathology scores in rAd26.RSV.F or rAd35.RSV.Fimmunized compared to rAd-e or mock immunized animals, as observed inthe prime-boost immunization experiments described above. Clearly,exacerbated disease was not observed, in contrast to animals that wereprimed with FI-RSV. Histopathology scores of animals immunized with rAdvectors were comparable to mock infected animals.

In conclusion, all single dose vaccination strategies resulted incomplete protection against RSV challenge replication, induced strongvirus neutralizing antibodies and did not show enhanced pathology.

Example 6

Vectors with variants such as fragments of RSV F or with alternativepromoters show similar immunogenicity.

The above examples have been conducted with vectors expressing thewild-type RSV F. Other, truncated or modified forms of F have beenconstructed in rAd35, providing embodiments of fragments of RSV F inadenoviral vectors. These truncated or modified forms of F include atruncated form of RSV-F wherein the cytoplasmic domain and transmembraneregion were lacking (i.e., only the ectodomain fragment remained), and afragment form of RSV-F with truncation of cytoplasmic domain andtransmembrane region and a further internal deletion in the ectodomainand addition of a trimerization domain. These vectors did not improvethe responses over rAd35.RSV.F with full-length F protein.

In addition, other rAd35 vectors with different alternative promotersdriving the expression of wild-type RSV F, have been constructed.

Immunogencity of the modified forms of RSV. F and the promoter variantshave been compared in the mouse model and compared to Ad35.RSV.F, whichexpress wild-type F. All Ad35 vectors harboring these F variants orpromoter variants showed responses in the same order of magnitude asAd35.RSV.F.

Example 7

Short term protection against RSV infection after recombinant adenoviralvectors immunization in vivo in a cotton rat model.

This experiment determines the potential of rapid onset of protection byadenoviral vectors expressing the RSV F protein in the cotton rat model.To this aim, cotton rats were immunized with a single i.m. injection of10⁷, 10⁸ or 10⁹ viral particles (vp) adenoviral vectors carrying thefull-length RSV F (A2) gene (Ad26.RSV.F) or no transgene (Ad26e) at day0 or at day 21. Animals infected intranasally with RSV A2 (10⁴ plaqueforming units (pfu)) were used as positive control for protectionagainst challenge replication, as it is known that primary infectionwith RSV virus protects against secondary challenge replication (Prince,Lab. Invest. 1999, 79:1385-1392). At day 49, seven or four weeks afterimmunization, the cotton rats were challenged intranasally with 1×10⁵pfu of plaque-purified RSV A2. Cotton rats were sacrificed 5 days afterinfection, a time point at which RSV challenge virus reaches peak titers(Prince, Lab. Invest. 1999, 79:1385-1392), and lung and nose RSV titerswere determined by virus plaque titration (Prince et al., 1978, Am. J.Pathology 93:711-791). FIG. 16 shows that high RSV virus titers in lungsand in nose were observed in animals receiving adenoviral vectorswithout transgene, respectively 4.8+/−0.11 log₁₀ pfu/gram and 5.1+/−0.32log₁₀ pfu/gram. In contrast, no challenge virus could be detected inlung and nose tissue from animals that received immunization withAd26.RSV.F vectors, independent of the time between immunization andchallenge. This experiment clearly indicates the rapid onset ofprotection against challenge virus replication by the Ad26 expressingRSV-F. Blood samples were taken from cotton rats immunized at day 0, atday 28 and at day of challenge (day 49). The sera were tested in aneutralization test for the induction of RSV specific antibodies (FIG.17). Immunization with adenoviral vectors induced dose-dependent VNAtiters. FIG. 18 shows that control animals do not have virusneutralizing antibodies at day 28 and day 49, while high VNA titers areinduced in animals 28 or 49 days after immunization with 10⁷ to 10⁹Ad26.RSV.F vp. Primary infection with RSV A2 virus resulted in rathermoderate VNA titers that gradually increased in time. This experimentclearly indicates the rapid onset of protection against challenge virusreplication by the Ad26 expressing RSV-F.

Example 8

Protection against RSV subgroup A and subgroup B infection afterrecombinant adenoviral vectors immunization in vivo in a cotton ratmodel.

RSV strains can be divided in two subgroup, the A and B subgroups. Thissubtyping is based on differences in the antigenicity of the highlyvariable G glycoprotein. The sequence of the F protein is highlyconserved but can also be classified in the same A and B subgroups.Patent application 0200 EO POO described that sera of Ad-RSV.F vectorsimmunized mice were also capable of cross-neutralizing the B1 strain invitro. FIG. 19 clearly shows that cotton rat serum derived from cottonrats immunized with Ad26.RSV-F_(A2) shows high VNA titers at day 49 postimmunization against RSV Long (subgroup A) and Bwash (subgroup B, ATCC#1540). The in vivo protection against either subgroup A or B challengewas determined in the cotton rat using low adenovirusvector doses of ina range from 10⁶ to 10⁸ vp. To this aim cotton rats were divided inexperimental groups of eight cotton rats each. Animals were immunized atday 0 by intramuscular injections of 10⁶, 10⁷, or 10⁸ viral particles(vp) adenoviral vectors carrying the full-length RSV F (A2) gene(Ad26.RSV.F) or no transgene (Ad26e) at day 0. At day 49, animals werei.n. challenged with either 10⁵ pfu RSV-A2, a RSV-A subgroup, or RSV-B15/97, a RSV-B strain. FIG. 20 shows that high RSV virus titers in lungsand in nose were observed in animals receiving adenoviral vectorswithout transgene. In contrast, no or limited challenge virus could bedetected in lung and nose tissue from animals that received immunizationwith Ad26.RSV.F. Only small differences were observed on protection whenchallenged with either RSV-A2 or RSV-B 15/97. Ad26.RSV.F_(A2) showedcomplete protection against lung challenge replication when using 10⁸and 10⁷vp doses, and exceptionally limited breakthrough at 10⁶ vpAd26.RSV.F_(A2). A similar trend was seen for protection against nosechallenge virus replication, although partial breakthrough was observedfor all animals at 10⁶ and 10⁷ vp Ad26.RSV.F_(A2), though lower than inthe control groups (FIG. 21). During the experiment, blood samples weretaken at day of challenge (day 49). The sera were tested in aneutralization test for the induction of RSV specific antibodies (FIG.22). This example demonstrates that adenoviral vectors at the low dosesof 10⁶ to 10⁸ vp Ad26.RSV showed a dose response of VNA titers againstRSV A2. Prior to immunization no virus neutralizing antibodies weredetected in any cotton rat.

Ad26.RSV.F proved to be somewhat better than Ad35.RSV.F, since thelatter showed some breakthrough in nose challenge experiments at a doseof 10⁸ vp.

Example 9

Protection against a high challenge dose of RSV-A2 after recombinantadenoviral vectors immunization in vivo in a cotton rat model.

This example determines the protection against a high challenge dose of5×10⁵ pfu compared to the standard dose of 1×10⁵ pfu RSV-A2. The studyenrolled cotton rats in experimental groups of eight cotton rats each.Animals were immunized by single intramuscular injections of 10⁷ or 10⁸viral particles (vp) adenoviral vectors carrying the full-length RSV F(A2) gene (Ad26.RSV.F) or no transgene (Ad26e) at day 0. Animalsinfected intranasally with RSV A2 (10⁴ plaque forming units (pfu)) wereused as positive control for protection against challenge replication.Cotton rats were sacrificed 5 days after infection, and lung and noseRSV titers were determined by virus plaque titration. FIG. 23 shows thata higher challenge dose induces higher lung viral load in animalsreceiving adenoviral vectors without transgene than with the standardchallenge dose. Animals that received immunization with 10⁷ or 10⁸ vpAd26.RSV.F vectors were completely protected against high and standardRSV challenge titers in the lungs. FIG. 24 shows that animals thatreceived immunization with 10⁸ vp Ad26.RSV.F vectors were completelyprotected against high and standard RSV challenge titers in the nose,while animals that received immunization with 10⁷ vp Ad26.RSV.F vectorswere partially protected against high and standard RSV challenge titers.

Example 10

Long term protection against RSV-A2 and RSV-B15/97 after recombinantadenoviral vectors immunization in vivo in a cotton rat model.

This example determines the durability of protection against RSV-A2 andRSV-B 15/97 after recombinant adenoviral vectors immunization in vivo ina cotton rat model. The study enrolled cotton rats in experimentalgroups of six cotton rats each. Animals were immunized by intramuscularinjections of 10⁸ viral particles (vp) or 10¹⁰ vp adenoviral vectorscarrying the full-length RSV F (A2) gene (Ad26.RSV.F) or no transgene(Ad26e or Ad35e). Animals were boosted 28 days later with the same vpdose, either with the same vector (Ad26.RSV.F) (homologous prime-boost)or with Ad35.RSV.F adenoviral (heterologous prime-boost); control groupswere immunized accordingly with Ad-e vectors, except that only one dosewas applied (10¹⁰). Some groups did not receive a booster immunization.Control groups consisted of six animals. Animals infected intranasallywith RSV A2 and B15/97 (10⁴ plaque forming units (pfu) were used aspositive control for protection against challenge replication. Challengewas at 210 days after the first immunization.

FIG. 25 shows that high RSV virus titers in lungs and in nose wereobserved in animals receiving adenoviral vectors without transgene. Incontrast, no challenge virus could be detected in lung tissue fromanimals that received immunization with Ad26.RSV.F and/or Ad35.RSV.F. NoRSV-A2 challenge virus could be detected in the nasal tissue fromanimals that received immunization with Ad26.RSV.F and/or Ad35.RSV.F.Challenge with RSV-B15/97 induced limited viral replication in the nasaltissues of animals that received immunization with Ad26.RSV.F and/orAd35.RSV.F, except for animals that received an Ad26.RSV.F primefollowed by an Ad35.RSV.F boost with 10¹⁰ vp. FIG. 26 shows the virusneutralizing antibody titers at 140 days post immunization. Adenoviralvector prime only or prime boost immunization with the doses of 10⁸ and10¹⁰ vp showed a dose response of VNA titers durable for at least 4.5months after immunization. Moreover the observed titers were higher thanthe neutralizing titers generated by primary i.n. immunization. A clearboost effect by either Ad26.RSV.F or Ad35.RSV.F on VNA titers wasobserved.

In conclusion, this example shows long lasting VNA titers afterimmunization with single or double doses of Ad26.RSV.F or Ad35.RSV.F,and long term full protection in lung and nose against homologous viruschallenge combined with long term full protection in lung and partialprotection in nose against heterologous virus challenge.

Example 11

Absence of vaccine-enhanced immunopathology after recombinant adenoviralvectors immunization in vivo in a cotton rat model.

To evaluate whether Ad26.RSV.F vaccine might exacerbate diseasefollowing a challenge with RSV A2, histopathological analyses of thelungs were performed 2 and 6 days after infection. Two days afterchallenge, the immediate response (including pulmonary neutrophilinfiltration) is peaking, whereas subacute changes such as lymphocyteinfiltration are peaking at day 6 post infection (Prince et al., J.Virol. 1986, 57:721-728). The study enrolled cotton rats in experimentalgroups of twelve cotton rats each. Animals were immunized byintramuscular injections of 10⁸ viral particles (vp) or 10¹⁰ vpadenoviral vectors carrying the full-length RSV F (A2) gene (Ad26.RSV.F)or no transgene (Ad26e). Some groups were boosted 28 days later with thesame vp dose with the same vector (Ad26.RSV.F) (homologous prime-boost);control groups were immunized accordingly with Ad-e vectors, except thatonly one dose was applied (10¹⁰). Control groups consisted of twelveanimals. Animals infected intranasally with RSV A2 (10⁴ plaque formingunits (pfu)) were used as positive control for protection againstchallenge replication. FI-RSV immunized animals were used as controlsfor enhanced disease. The lungs were harvested, perfused with formalin,sectioned, and stained with hematoxylin and eosin for histologicexamination. Histopathology score was done blinded, according tocriteria published by Prince (Prince et al., Lab. Invest. 1999,79:1385-1392), and scored for the following parameters:peribronchiolitis, perivasculitis, interstitial pneumonitis, andalveolitis. The scoring of the lung pathology of this experiment isdepicted in FIG. 27 for day 2 and in FIG. 28 for day 6. Following RSVchallenge, FI-RSV immunized animals showed at day 2 and day 6 elevatedhistopathology on all histopathology parameters examined compared tomock-immunized and challenged animals, which was expected based onearlier published studies. Histopathology scores in all groups immunizedwith Ad26.RSV.F vectors were comparable to the mock immunized animals atday 2 and were at day 6 post challenge always scored lower than themock-immunized challenged (Ad26.e). Thus, the Ad26.RSV.F vaccines didnot result in enhanced disease, unlike FI-RSV vaccines.

Example 12

Ad26.RSV.F prime boosted with recombinant F protein results in a Th1skewed response in a mouse model.

In this example, it was investigated whether the immune response uponAd26.RSV.F prime can be enhanced by boosting with adjuvanted recombinantRSV F protein. To this aim mice were divided in experimental groups ofseven mice each. Animals were immunized at day 0 by intramuscularinjections of 10¹⁰ viral particles (vp) adenoviral vectors carrying thefull-length RSV F (A2) gene (Ad26.RSV.F) or PBS. At day 28, animals wereboosted i.m. with either the same vector in the same dose, or withadjuvanted RSV F protein (full-length; post-fusion conformation: post-F)(in 2 doses: 5 μg and 0.5 μg). FIG. 29 clearly shows that serum derivedfrom mice immunized with Ad26.RSV-F_(A2) and boosted with adjuvanted RSVF shows high VNA titers at 12 weeks post immunization against RSV-A Long(subgroup A). FIG. 30 shows the IgG2a/IgG1 ratio in the sera of miceimmunized with Ad26.RSV-F_(A2) and boosted with adjuvanted RSV Fprotein. A high ratio is indicative of a Th1 balanced responses, whereasa low ratio indicates a Th2 skewed response. Clearly, Ad26.RSV.Fimmunized animals, boosted with either Ad26.RSV.F or RSV F proteinresults in a high IgG2a/IgG1 ratio, whereas control mice immunized withFI-RSV or RSV F protein (without the context of adenoviral vectors)induce a low ratio. Because a Th1 skewed response is strongly desired inan RSV vaccine to avoid enhanced disease upon challenge and to inducestrong T cell memory, the Th2 skewing response of a protein immunizationcan be directed towards a Th1 response when an Ad26.RSV.F prime isapplied. FIG. 31 shows the cellular responses in spleens derived frommice immunized with Ad26.RSV-F_(A2) and boosted with adjuvanted RSV Fprotein. It can clearly be observed that boosting with adjuvanted RSV Fprotein will strongly increase the cellular response as well.

1. A composition for vaccinating a subject against respiratory syncytialvirus (RSV) F protein, composition comprising: a recombinant humanadenovirus of serotype 26 that comprises a nucleic acid moleculeencoding an RSV F protein or a fragment of an RSV F protein, whichcomposition, upon a single intramuscular injection to a Cotton rat(Sigmodon hispidus), gives complete protection against RSV challengereplication in a Cotton rat model of respiratory viral infection.
 2. Thecomposition of claim 1, wherein the nucleic acid molecule encodes apeptide comprising SEQ ID NO:
 1. 3. The composition of claim 1, whereinthe nucleic acid molecule encoding RSV F protein has been codonoptimized for expression in a human cell.
 4. (canceled)
 5. Thecomposition of claim 1, wherein the recombinant human adenovirus has: adeletion in the E1 region of the adenoviral genome, a deletion in the E3region of the adenoviral genome, or a deletion in each of the E1 and theE3 regions of the adenoviral genome.
 6. The composition of claim 1,wherein the recombinant adenovirus has a genome comprising, at its 5′end, CTATCTAT.
 7. A method for vaccinating a subject against respiratorysyncytial virus (RSV), the method comprising: administering to thesubject the composition of claim
 1. 8. The method according to claim 7,wherein the composition is administered to the subject intramuscularly.9. The method according to claim 7, wherein the composition isadministered to the subject in a single administration.
 10. The methodaccording to claim 7, further comprising: administering to the subjectrecombinant human adenovirus of serotype 35 that comprises a nucleicacid encoding an RSV F protein or fragment thereof.
 11. A method ofimmunizing a subject against respiratory synctial virus (RSV), themethod comprising: intramuscularly administering the composition ofclaim 1 to the subject in a single administration.
 12. The methodaccording to claim 7, further comprising: administering RSV F protein tothe subject.
 13. A method for reducing infection and/or replication ofrespiratory syncytial virus (RSV) in a subject, the method comprising:administering to the subject, by intramuscular injection, a compositioncomprising a recombinant human adenovirus of serotype 26 comprising anucleic acid molecule encoding a respiratory syncytial virus (RSV) Fprotein or fragment thereof, which composition, upon a singleintramuscular injection, gives complete protection against RSV challengereplication in a Cotton rat (Sigmodon hispidus) model of respiratoryviral infection.
 14. A host cell comprising: a recombinant humanadenovirus of serotype 26 comprising a nucleic acid molecule encoding arespiratory syncytial virus (RSV) F protein or fragment thereof, whichrecombinant human adenovirus upon a single intramuscular injection to aCotton rat (Sigmodon hispidus), gives complete protection against RSVchallenge replication in a Cotton rat (S. hispidus) model of respiratoryviral infection.
 15. A process for producing the composition of claim 1,the process comprising: providing a recombinant human adenovirus ofserotype 26 that comprises a polynucleotide encoding an RSV F protein orfragment thereof, propagating the recombinant human adenovirus in aculture of host cells, isolating and purifying the recombinant humanadenovirus therefrom, and formulating the purified recombinant humanadenovirus to form the composition.
 16. A polynucleotide encoding arecombinant human adenovirus of serotype 26 that comprises a nucleicacid molecule encoding a respiratory syncytial virus (RSV) F protein ora fragment thereof, wherein the recombinant human adenovirus after beingexpressed from said polynucleotide, upon a single intramuscularinjection to a Cotton rat (Sigmodon hispidus), gives complete protectionagainst RSV challenge replication in a Cotton rat (S. hispidus) model ofrespiratory viral infection.
 17. The method according to claim 11,further comprising: administering to the subject recombinant humanadenovirus of serotype 35 that comprises a nucleic acid moleculeencoding an RSV F protein or fragment thereof.
 18. The method accordingto claim 8, wherein the composition is administered to the subject in asingle administration.
 19. The method according to claim 18, furthercomprising: administering to the subject recombinant human adenovirus ofserotype 35 that comprises a nucleic acid encoding an RSV F protein orfragment thereof.
 20. The method according to claim 8, furthercomprising: administering RSV F protein to the subject.
 21. The methodaccording to claim 9, further comprising: administering RSV F protein tothe subject.